Immunofluorescence-digital image processing system for the quantitation of secreted immunoglobulin by single cells

To quantitate the amount of secreted immunoglobulin (Ig) by a single cell, the immunofluorescence digital image processing (IDIP) system was adapted to the modified enzyme-linked immunospot (ELI-SPOT) assay. In this assay, an immunofluorescence (tetramethylrhodamine isothiocyanate) conjugated antibody was used for the detection of spots instead of the usual method of enzyme coupling. We have named this the immunofluorescence-linked immunospot (ILISPOT) assay. In addition to the quantitation of secreted Ig by single cells, this method allowed us to objectively determine the exact number of Ig producing spot forming cells (SFC). 96 well culture plates were pre-coated with goat anti-mouse Ig. The mouse IgM producing hybridoma (E-3-4) was incubated in the plates for 4 h at 37 degrees C. Cells were removed prior to the addition of biotinylated goat anti-mouse mu antibody. After overnight incubation, immunofluorescence conjugated avidin was added for the visualization of spots by the IDIP system. The IDIP system consists of a fluorescent microscope equipped with a video camera and computer. The gray scale of secreted IgM was initially established as a standard by the known amount of purified IgM. By using digital image processing, the number of spots and the gray scale of individual spots were computed. The shape and pattern of gray scale data were used to distinguish between the real spots and pseudo spots. This IDIP system could detect as little as 0.19 pg of secreted IgM (1.2 x 10(5) molecules) and an average of approximately 1.33 pg (8.3 x 10(5) molecules) produced by a single cell. Adaptation of the digital image processing system to the ILISPOT assay allowed the measurement of both the amount of Ig produced at the single cell level and also the exact numbers of SFC present in a totally objective fashion.